Food Microbiology 44 (2014) 1e5
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Food Microbiology
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Antimicrobial activity of reuterin produced by Lactobacillus reuteri on Listeria monocytogenes in cold-smoked salmon
R. Montiel a, I. Martín-Cabrejas a, S. Langa a, N. El Aouad b, J.L. Arques a, F. Reyes b, * M. Medina a, a Departamento Tecnología de Alimentos, INIA, Carretera de La Coruna~ Km 7, Madrid 28040, Spain b Fundacion MEDINA, Avenida del Conocimiento 3, Parque Tecnologico de Ciencias de la Salud, Granada 18016, Spain article info abstract
Article history: Lactobacillus reuteri INIA P579 was used for the production and purification of reuterin. The purity of Received 8 January 2014 reuterin was assessed by high resolution electrospray ionization mass spectrometry (HRESIMS) and Received in revised form nuclear magnetic resonance (NMR) spectroscopy. After purification, reuterin concentration obtained was 9 May 2014 1.3 M. The inhibitory activity using Escherichia coli K12 as indicator strain was estimated to be 510 AU/ml. Accepted 14 May 2014 Survival curves in tryptic soy broth revealed that reuterin required to inhibit the growth of three Listeria Available online 22 May 2014 monocytogenes strains was in the range of 2e4 AU/ml. Purified reuterin (10 AU/g) significantly reduced the growth of L. monocytogenes in cold-smoked salmon kept under moderate or strong temperature Keywords: abuse conditions. After 15 d at 8 C, cold-smoked salmon with added reuterin exhibited L. monocytogenes Reuterin L. monocytogenes counts 2.0 log CFU/g lower than control smoked salmon with no reuterin added. At 30 C, reuterin also Cold-smoked salmon controlled the growth of the pathogen, with counts 1.4 and 0.9 log CFU/g lower than those observed in control smoked salmon after 24 and 48 h, respectively. The addition of purified reuterin might be used as a hurdle technology to improve the safety and extend the shelf-life of lightly preserved seafood products such as cold-smoked salmon. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction products has received increased interest in recent decades. The addition of antimicrobial substances produced by LAB to control Listeria monocytogenes is a Gram-positive microorganism able to pathogen bacteria has mainly focused on the application of bacte- grow under different stress conditions such as refrigeration tem- riocins (nisin, pediocin or divergicin) in milk and dairy products peratures, low pH or high levels of NaCl, which is frequently (Arques et al., 2008a,b), vegetables (Huang et al., 2009), meat detected in cold-smoked salmon and in the environment of pro- products (Nieto-Lozano et al., 2010) or seafood products (Szabo and cessing plants (Lakshmanan and Dalgaard, 2004; Warriner and Cahill, 1999; Tahiri et al., 2009), whereas studies about the appli- Namvar, 2009). L. monocytogenes is a great concern for the manu- cation of non-bacteriocin antimicrobial substances as reuterin are facturers of cold-smoked salmon as the combination of salt and limited. temperature used is not enough to inhibit the growth of this Reuterin or b-hydroxypropionaldehyde (b-HPA) is an interme- pathogen. Additional hurdles are needed to reduce or eliminate its diate compound produced by some strains of Lactobacillus reuteri presence in this slightly processed seafood product. Although during the anaerobic metabolism of glycerol (Talarico and L. monocytogenes was seldom detected above the legal limit of Dobrogosz, 1989). Reuterin is active towards a broad spectrum of safety from ready-to-eat foods at retail level in the European Union, food-borne pathogens and spoilage microorganisms, is soluble in samples exceeding this limit (100 CFU/g) were most often found in water, resistant to proteolytic and lipolytic enzymes and stable over fishery products and fermented sausages (EFSA, 2013). a wide range of pH (El-Ziney et al., 2000). Therefore, it has been Biopreservation by lactic acid bacteria (LAB) or their metabolites proposed to improve the safety and quality of foods, reducing the to improve the safety and to extend the shelf-life of different food addition of chemical preservatives (Axelsson et al., 1989; Vollenweider et al., 2003). In aqueous solution, reuterin is a dy- namic system constituted by three molecules, 3- hydroxypropionaldehyde (3-HPA), 1,1,3-trihydroxypropane (HPA * þ þ Corresponding author. Tel.: 34 913476774; fax: 34 913572293. hydrate) and 2-(2-hydroxy-ethyl)-4-hydroxy-1,3-dioxane (HPA E-mail address: [email protected] (M. Medina). http://dx.doi.org/10.1016/j.fm.2014.05.006 0740-0020/© 2014 Elsevier Ltd. All rights reserved. 2 R. Montiel et al. / Food Microbiology 44 (2014) 1e5 dimer). The mode of action is not fully understood, but recent The purity of reuterin in samples was assessed by high resolu- studies have demonstrated that the aldehyde group of 3-HPA is tion electrospray ionization mass spectrometry (HRESIMS) per- mainly responsible for the antimicrobial activity. Reuterin reacts formed on a Bruker maXis QTOF spectrometer, and Nuclear with sulfhydryl groups of proteins and small molecules inducing Magnetic Resonance (NMR) spectroscopy acquired on a Bruker oxidative stress responses (Schaefer et al., 2010; Vollenweider et al., AVANCE III 500-MHz NMR spectrometer equipped a with a 1.7 mm 2010). The antimicrobial activity of reuterin against Gram-positive TCI cryoprobe. The mass spectrometer was operated in positive ESI and Gram-negative pathogens has been reported in milk and dairy mode. The instrumental parameters were: 4 kV capillarity voltage, products (El-Ziney and Debevere, 1998; Arques et al., 2008a,b; drying gas flow of 1 L/min at 200 C, nebulizer pressure at 2.8 bars. Arques et al., 2011) and in meat products (El-Ziney et al., 1999; TFA-Na cluster ions were used for mass calibration of the instru- Kuleas¸ an and Çakmakçi, 2002). However, the use of reuterin to ment prior to samples injection. Pre-run calibration was by infusion control food-borne pathogens has not been applied in fishery with the same TFA-Na calibrant. NMR spectra (500 and 125 MHz for 1 13 products before and could be an interesting approach to control H and C NMR, respectively) were recorded in CD3OD, using the L. monocytogenes in cold-smoked salmon. signals of the residual solvent as internal references (dH 3.31 and dC The aim of the present work was to purify reuterin produced by 49.0 ppm). The purified reuterin without contaminants was diluted L. reuteri INIA P579 and to evaluate its antimicrobial activity against in sterile distilled water to about 100 mg/ml solution and stored at L. monocytogenes in cold-smoked salmon under conditions of 40 C until use. moderate and strong temperature abuse. 2.3. Quantification of the concentration and inhibitory activity of 2. Materials and methods reuterin
2.1. Bacteria growth conditions Reuterin concentration was quantified by a colorimetric method adapted from Circle et al. (1945). Briefly, serial dilutions of purified L. reuteri INIA P579 (from the INIA culture collection, Instituto reuterin in sterile distilled water were prepared and colorimetric Nacional de Investigacion y Tecnología Agraria y Alimentaria, reaction after the addition of tryptophan solution (0.01 M solution Madrid, Spain) (Langa et al., 2013) was used for reuterin production. in 0.1 N HCl) and concentrated HCl (32%, 10 N) were carried out. L. monocytogenes strains INIA H66a and INIA 2530 isolated from Solutions were incubated at 60 C for 5 min in a Thermomixer industrial environment, L. monocytogenes CECT 5725 and Escher- compact (Eppendorf, Hamburg, Germany) to develop colour. The ichia coli K12 CECT 443 (from the Spanish Type Culture Collection, absorbance was measured at 490 nm with a Beckman DU-650 Valencia, Spain) were used as test organisms in this study. The spectrophotometer (Beckman Coulter, Inc., Palo Alto, CA, USA). A strains were maintained as stock culture at 80 C in Trypticase Soy standard curve of acrolein in water (0e4 mM) was also prepared Yeast Extract Broth (TSYEB, Biolife s.r.l., Milano, Italy) supplemented plotting acrolein concentrations against the average of three with 30% glycerol. absorbance measurements for each concentration. Samples with reuterin were compared against the acrolein standard to calculate 2.2. Production and purification of reuterin molar concentration of reuterin stock solution. Experiments were carried out in triplicate. The reuterin-producing L. reuteri was subcultured anaerobically The inhibitory activity of reuterin stock solution was determined (AnaeroGen™, Oxoid Ltd., Basingstoke, Hampshire, UK) in MRS broth by using the modified assay of Chung et al. (1989) using E. coli K12 (Biolife) at 37 C for 18 h before use in experiments. The bacterial as indicator strain. Reuterin aqueous solution was serially diluted in strain was inoculated in 2-L MRS broth flasks and incubated for 18 h sterile water and 150 ml were added to microtiter plate wells. at 37 C under anaerobic conditions. Cells were harvested by Overnight culture of test strain was diluted to approximately centrifugation at 4500e5000 g for 5 min and washed once with 104 CFU/g in double strength Tryptic Soy Broth (TSB, Biolife) and sterile glycerol solution (100 mM). The pellet was resuspended in a 150 ml volumes were dispensed to each well. Distilled water total volume of 500 ml of 100 mM glycerol solution and incubated for without reuterin was used as negative control. Plates were incu- 3hat37 C under anaerobic conditions. Cells were removed by bated at 37 C and the absorbance was read during 24 h using a centrifugation at 6000 g for 5 min and the supernatant was Multiskan Spectrum microplate reader (Multiskan Spectrum, transferred into new flasks and frozen at 20 C until its purification. Thermo Labsystems, Finland) at a wavelength of 600 nm. Reuterin Reuterin purification was performed using the method arbitrary units (AU) were defined as the reciprocal of the highest described by Vollenweider et al. (2003) with some changes. Briefly, two-fold dilution that did not allow the growth of the indicator 250 ml of the solution containing reuterin were frozen under strain. Three independent experiments were conducted and three rotation in a MeOH bath ( 30 C) and lyophilized for 16e20 h. To replicates per reuterin concentration were analyzed. eliminate the orange-colored impurities, the viscous liquid was resuspended with 40 ml of acetone (Merck, Darmstadt, Germany), 2.4. Antimicrobial activity on L. monocytogenes mixed with 10 g of silica gel 60 (Merck), evaporated and poured in a glass Buchner funnel with fritted disc containing 5 g of silica gel 60. The inhibitory activity of reuterin against 3 strains of The reuterin was eluted with acetone:ethyl acetate (2:1) in 100 ml L. monocytogenes was determined by measuring the optical density glass flasks and detected using the colorimetric method described (OD) in an automated spectrophotometer (Bioscreen C, Bio-Rad by Rahn and Schlenk (1973) for detection of aldehydes. Eluted Laboratories S.A., Madrid, Spain). Reuterin was diluted in sterile fractions containing reuterin were concentrated under reduced distilled water and 150 ml were added to microtiter plate wells to pressure, loaded on a silica gel 60 column and eluted with aceto- achieve a final concentration of 0.5, 1, 2, 4, 6, 8 and 10 AU/ml. ne:ethyl acetate (2:1), using a CombiFlash RF4x chromatography Overnight cultures at approximately 106 CFU/ml were inoculated in system (Teledyne Isco, Inc., Massachusetts, USA) and a flow rate of double strength Tryptic Soy Broth (TSB, Biolife) and 150 ml volumes 5 ml/min. Fractions with the aldehyde indicated by the colorimetric were dispensed to each well. Distilled water without reuterin was method cited above were combined and evaporated under reduce used as negative control. Microwell plates were incubated at 37 C pressure and dried in a vacuum system to remove last traces of for 24 h, and optical density at 600 nm was determined at 30 min solvents. intervals. DMFit Excel add-in software (kindly provided by Dr. J. R. Montiel et al. / Food Microbiology 44 (2014) 1e5 3
Baranyi, Institute of Food Research, Norwich, UK) was used to fitthe 0.70 experimental growth data, estimating the kinetic parameters, lag a time (l, expressed in hours) and maximum growth rate (m, 0.60 expressed in OD/h). Three independent experiments were con- ducted and three replicates per reuterin concentration were 0.50 analyzed. 0.40
2.5. Preparation of smoked salmon samples 0.30 OD 600nm OD
Cold-smoked salmon (Salmo salar, L.) was purchased from a 0.20 local supermarket. Samples were cut into 20 g pieces and inocu- lated by adding 100 ml of an overnight culture of L. monocytogenes 0.10 INIA 2530 to achieve a final population of approximately 105 CFU/g. An aqueous solution of purified reuterin (100 mg/ml) was added on 0.00 0 3 5 8 10 13 15 18 20 23 25 the surface of smoked salmon to reach an estimated final activity of 10 AU/g. Smoked salmon that had not been treated with reuterin time (h) was used as control. Samples were maintained at 8 C and 30 C for 0.70 15 d and 48 h, respectively. Two trials with duplicate samples were b carried out for each temperature assayed. 0.60
2.6. Microbiological analysis 0.50
Salmon slices were diluted 10-fold with sterile 0.1% (wt/vol) 0.40 peptone water solution and homogenized with a Stomacher 400 0.30
(A.J. Seward Ltd., London, UK). Decimal dilutions prepared in the 600nm OD same solution, spread on duplicated plates of PALCAM Listeria Se- 0.20 lective Agar (Merck, Darmstadt, Germany) and incubated at 37 C for 48 h. 0.10
2.7. Statistical analysis 0.00 0 3 5 8 10 13 15 18 20 23 25
Analysis of variance with treatment, temperature of storage and time (h) time of refrigeration as main effects was carried out using SPSS Win 12.0 software (SPSS Inc., Chicago, IL, USA). The significance of dif- 0.70 ferences between means for the same time of storage was evalu- c ated by Tukey test with a confidence interval of 95%. 0.60
0.50 3. Results and discussion 0.40 3.1. Reuterin concentration 0.30 About 0.73 g of pure reuterin was obtained per litre of glycerol 600nm OD solution processed. The purity of the reuterin, colourless and highly 0.20 viscous, was verified with HRESIMS and NMR and only fractions 0.10 without contaminants, including glycerol or 1,3-propanediol, were used in the subsequent experiments. A work solution of 100 mg/ml 0.00 of reuterin was prepared and stored at 40 C until use. The con- 0 3 5 8 10 13 15 18 20 23 25 centration of reuterin in the stock solution was estimated to be time (h) 1.3 M. Fig. 1. Survival curves of L. monocytogenes INIA 2530 (a), L. monocytogenes H66a (b) 3.2. Inhibitory activity and L. monocytogenes CECT 5725 (c) at 37 C in double strength TSB with purified reuterin [0.5 AU/ml (B), 1 AU/ml (:), 2 AU/ml ( ), 4 AU/ml(þ) and control without reuterin (A)]. The inhibitory activity of reuterin, determined using E. coli K12 as indicator strain, was estimated to be 510 AU/ml. The antimi- crobial effect of reuterin on L. monocytogenes growth was deter- L. monocytogenes INIA 2530 was the most resistant strain to reu- mined by measuring the optical density. Survival curves of the terin growing at 2 AU/ml after 20.43 h, and was chosen as test strain three strains studied in double strength TSB with reuterin at for the experiments in cold-smoked salmon. different concentrations are shown in Fig. 1. Estimated maximum growth rates and lag time by using DMFit Excel add-in software are shown in Table 1. For the three strains, maximum growth rates 3.3. Effect of reuterin on L. monocytogenes INIA 2530 in cold- were significantly reduced (P < 0.001) and lag times increased smoked salmon (P < 0.001) by reuterin. According to the data obtained in the microplate reader, the concentration required to inhibit the growth The effect of reuterin at 10 AU/g on the growth of of L. monocytogenes strains INIA 2530, INIA H66a and CECT 5725 L. monocytogenes in cold-smoked salmon at 8 and 30 C is shown in during 24 h at 37 C was 4, 4, and 2 AU/ml, respectively. Figs. 2 and 3, respectively. According to the analysis of variance, 4 R. Montiel et al. / Food Microbiology 44 (2014) 1e5
Table 1 Estimated maximum growth rates and lag times of L. monocytogenes in the presence of different concentrations of reuterin.
Reuterin AU/ml L. monocytogenes INIA 2530 L. monocytogenes INIA H66a L. monocytogenes CECT 5725